Chelator conjugates

ABSTRACT

The present invention relates to improved chelator conjugates with biological targeting molecules, suitable for forming metal complexes with radiometals. The radiometal complexes, especially with the radiometal 99m Tc, are useful as radiopharmaceuticals.

This application is a filing under 35 U.S.C. § 371 and claims priorityto international application number PCT/GB02/03168 filed Jul. 10, 2002which application claims priority to application number 0116815.2 filedon Jul. 10, 2001, in Great Britain, the entire disclosure of which isincorporated therein.

FIELD OF THE INVENTION

The present invention relates to improved chelator conjugates withbiological targeting molecules, suitable for forming metal complexeswith radiometals. The radiometals. The radiometal complexes are usefulas radiopharmaceuticals, especially with ^(99m)Tc.

BACKGROUND TO THE INVENTION

Diaminedioximes are a known class of chelating agents, which have beenshown to form

-   -   Q=—(CH₂)₃— ie. propyleneamine oxime or PnAO;    -   Q=—(CH₂)₄— ie. butyleneamine oxime or BnAO;    -   Q=—(CH₂)₅— ie. pentyleneamine oxime or PentAO;        complexes with the radiometal ^(99m)Tc.

The ligand PentAO was first disclosed by S. Jurisson et al [Inorg. Chem,26, 3576-82 (1987], who showed that its' metal complex with thelong-lived radiometal ⁹⁹Tc was neutral, with a Tc(V) dioxo core (ie.TcO₂ ⁺). J-M Lo et al [Appl. Rad. Inst, 44, 1139-46 (1993)] describedthe synthesis of PentAO and it's complexation with ^(99m)Tc.

U.S. Pat. No. 5,688,487 discloses chelate-conjugates of diaminedioximeshaving a C₂₋₅ alkylene bridge with nitroimidazole biological targetingmolecules, for hypoxia imaging. Conjugation of the nitroimidazole at theC1 (oxime methyl) position is described.

WO 95/04552 discloses nitroimidazole conjugates of BnAO and PentAO. TheExample shows conjugation at the C1 (oxime methyl) position

WO 95/19187 discloses conjugates of linear or cyclic 3-50 mer syntheticpeptides with polydentate chelating agents attached at the peptidecarboxyl terminus, for use as radiopharmaceuticals. Diaminedioximes suchas PnAO, BnAO and PentAO are described as suitable chelating agents.

WO 99/60018 discloses diaminedioxime chelate conjugates ofdiaminedioxime ligands with peptides for thrombus imaging. A preferredsuch chelator is said to be a diaminedioxime with Q=—(CH₂)₂NR(CH₂)₂—.

THE PRESENT INVENTION

The diaminedioxime-peptide chelator conjugates of WO 99/60018 of FormulaI:

-   -   where T=N and Y=—CH₂CH₂NH-[peptide],        do, however, suffer from significant disadvantages. Thus, on        chelation with ^(99m)Tc, this aza-diaminedioxime forms several        technetium species, which can be separated and detected by        chromatography. At ambient temperature, the initial        radiolabelled species (intermediates) are converted over time        (2-3 hrs) to a stable product. This intermediate-product        conversion can be promoted by the use of higher pH (>pH 8) and        heating. These conditions are not ideal in a hospital        radiopharmacy, therefore a chelator with fewer intermediates        and/or a faster intermediate-product conversion rate is        desirable. Clearly, the need for heating and possibly relatively        high pH to achieve adequate radiochemical purity (RCP) of the        desired ^(99m)Tc species is undesirable, since such heating may        degrade the attached biological targeting molecule or peptide. A        further problem with the aza-diaminedioxime chelators of Formula        I is that the tertiary amine nitrogen of the bridgehead position        is relatively basic. This means that on formation of the        corresponding ^(99m)Tc complex in aqueous solution, the tertiary        amine is at least partially protonated, with the result that the        conjugate is charged. This charge may limit the applications of        the labelled biological targeting moiety, since the charge may        make it more difficult for the radiolabelled conjugate to cross        cell membranes.

The present invention provides an alternative chelator system (Formula Iwhere T=C), which overcomes these prior art problems, and providesconjugates which can be radiolabelled to give good RCP at roomtemperature, under aqueous conditions at near neutral pH. The radiometalcomplexes are of good stability. Prior art N2S2 and N3S thiol-containingbifunctional chelators suffer from the disadvantage that the thiols areair sensitive, rapidly oxidising in air to the corresponding disulphidesunder neutral to basic conditions. They must therefore be kept in aninert atmosphere before use or in a protective matrix. Alternativelythey can be used as protected species such as thioacetate, or atetrahydropyranyl hemithioketal but this necessitates removal ofprotecting groups before use with acid or base and heating. All thesefeatures reduce the convenience of these chelators compared to thechelators of the present invention. Hence, the present chelators areuseful for the conjugation and radiolabelling of a wide range ofbiological targeting moieties

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the present invention provides, a chelator conjugateof a diaminedioxime ligand with a biological targeting moiety. The term“chelator conjugate” means a compound where a metal chelating agent iscovalently linked (‘conjugated’) to a biological targeting moiety. Thechelator conjugate is of Formula II:

-   -   where:    -   each R¹, R² and R³ is independently an R group;    -   Y is —(A)_(n)—X-Z        -   where: X is —NR⁴—, —CO₂—, —N(C═S)—, —N(C═O)—, —S— or —O—;            -   Z is a biological targeting moiety,            -   R⁴ is independently an R group;            -   -(A)_(a)- is a linker group where each A is                independently —CR₂—, —CR═CR—, —C═C—, —NRCO—, —CONR—,                —SO₂NR—, —NRSO₂—, —CR₂OCR₂—, —CR₂SCR₂—, —CR₂NRCR₂—, a                C₄₋₈ cycloheteroalkylene group, a C₄₋₈ cycloalkylene                group, a C₅₋₁₂ arylene group, a C₅₋₁₂ heteroarylene                group or a polyalkyleneglycol, polylactic acid or                polyglycolic acid moiety;            -   n is an integer of value 0 to 10;    -   each R group is independently H or C₁₋₁₀ alkyl, C₃₋₁₀ alkyaryl,        C₂₋₁₀ alkoxyalkyl, C₁₋₁₀ hydroxyalkyl, C₁₋₁₀ fluoroalkyl or 2 or        more R groups, together with the atoms to which they are        attached form a carbocyclic, heterocyclic, saturated or        unsaturated ring.

By the term “biological targeting moiety” is meant: 3-100 mer peptidesor peptide analogues which may be linear peptides or cyclic peptides orcombinations thereof; monoclonal antibodies or fragments thereof; orenzyme substrates or inhibitors; synthetic receptor-binding compounds;oligonucleotides, or oligo-DNA or oligo-RNA fragments. The biologicaltargeting moiety may be of synthetic or natural origin, but ispreferably synthetic. Preferred biological targeting moieties are 3-20mer peptides, which may be of synthetic or natural origin, but arepreferbly synthetic. By the term “cyclic peptide” is meant a sequence of5 to 15 amino acids in which the two terminal amino acids are bondedtogeter by a covalent bond which may be a peptide or disulphide bond ora synthetic non-peptide bond such as a thioether, phosphodiester,disiloxane or urethane bond.

By the term “amino acid” is meant an L- or D-amino acid, amino acidanalogue or amino acid mimetic which may be naturally occurring or ofpurely synthetic origin, and may be optically pure, i.e. a singleenantiomer and hence chiral, or a mixture of enantiomers. Preferably theamino acids of the present invention are optically pure. By the term“amino acid mimetic” is meant synthetic analogues of naturally occurringamino acids which are isosteres, i.e have been designed to mimic thesteric and electronic structure of the natural compound. Such isosteresare well known to those skilled in the art and include but are notlimited to depsipeptide, retro-inverso peptides, thiodes, cycloalkanesor 1,5-disubstituted tetazoles [see M. Goodman, Biopolymers, 24, 137,(1985)].

Suitable peptides for use in the present invention include:

-   -   somatostatin, octreotide and analogues,    -   peptides which bind to the ST receptor, where ST refers to the        heat-stable toxin produced by E.coli and other micro-organisms;    -   laminin fragments eg. YIGSR, PDSGR, IKVAV, LRE and        KCQAGTFALRGDPQG,    -   N-formyl peptides for targeting sites of leucocyte accumulation,    -   Platelet factor 4 (PF4) and fragments thereof;    -   RGD-containing peptides,    -   peptide fragments of α₂-antiplasmin, fibronectin or beta-casein,        fibrinogen or thrombospondin. The amino acid sequences of        α₂-antiplasmin, fibronectin, beta-casein, fibrinogen and        thrombospondin can be found in the following references:        α₂-antiplasmin precursor [M. Tone et al., J.Biochem, 102, 1033,        (1987)]; beta-casein [L. Hansson et al. Gene, 139, 193, (1994)];        fibronectin [A. Gutman et al, FEBS Lett., 207, 145, (1996)];        thrombospondin-1 precursor [V. Dixit et al, Proc. Natl. Acad.        Sci., USA, 83, 5449, (1986)]; R. F. Doolittle, Ann. Rev.        Biochem., 53, 195, (1984).

Preferably the peptides of the present invention comprise an amino acidsequence is taken from the N-terminus of:

-   (i) α₂-antiplasmin,-   i.e. NH₂-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-OH    or variants of this in which one or more amino acids have been    exchanged, added or removed such as:-   NH₂-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-Gly-OH,-   NH₂-Asn-Gln-Glu-Ala-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-Gly-OH,-   NH₂-Asn-Gln-Glu-Gln-Val-Gly-OH; or-   (ii) casein-   ie. Ac-Leu-Gly-Pro-Gly-Gln-Ser-Lys-Val-Ile-Gly.

Synthetic peptides of the present invention may be obtained byconventional solid phase synthesis, as described in P. Lloyd-Williams,F. Albericio and E. Girald; Chemical Approaches to the Synthesis ofPeptides and Proteins, CRC Press, 1997.

Suitable monoclonal antibodies or fragments thereof for use in thepresent invention include: antibodies to the CD-20 antigen expressed onthe surface of B-cells; anti-leucocyte or anti-granulocyte antibodies;anti-myosin antibodies or antibodies to carcinoembryonic antigen (CEA).

Suitable enzyme substrates or inhibitors include glucose and glucoseanalogues such as fluorodeoxyglucose; fatty acids or elastaseinhibitors.

Suitable synthetic receptor-binding compounds include estradiol,estrogen, progestin, progesterone and other steroid hormones; ligandsfor the dopamine D-1 or D-2 receptor, or dopamine transporter such astropanes; and ligands for the serotonin receptor.

By the term ‘fluoroalkyl’ is meant an alkyl group with at least onefluorine substituent, ie. the term encompasses groups frommonofluoroalkyl (eg. —CH₂F) to perfluoroalkyl (eg. CF₃).

In the diaminedioxime chelators of the present invention, R³ ispreferably H. It is also preferred that at least one R² group is H₁ morepreferably all the R² groups are H. Each R¹ is preferably C₁₋₃ alkyl,C₂₋₄ alkoxyalkyl, C₁₋₃ hydroxyalkyl, or C₁₋₃ fluoroalkyl, and is mostpreferably C₁₋₃ alkyl or C₁₋₃ fluoroalkyl. It is most especiallypreferred that all the R¹ groups are CH₃₋

Preferred chelator conjugates of Formula II wherein 2 or more R groupswhich, together with the atoms to which they are attached, form acarbocyclic, heterocyclic, saturated or unsaturated ring, comprise suchrings having 3- to 6-members, especially 5- or 6-member. Most preferredsuch rings are saturated carbocyclic rings. Preferred carbocyclic ringsare those in which 2 R¹ groups attached to either the same or adjacentcarbon atoms are combined to form 3- to 6-membered, especially 5- or6-membered saturated rings.

It is envisaged that the role of the linker group -(A)_(n) is todistance the relatively bulky radiometal complex which results uponmetal coordination, from the active site of the biological targetingmoiety so that eg. receptor binding is not impaired. This can beachieved by a combination of flexibility (eg. simple alkyl chains), sothat the bulky group has the freedom to position itself away from theactive site and/or rigidity such as a cycloalkyl or aryl spacer whichorientates the metal complex away from the active site. The nature ofthe linker group can also be used to modify the biodistribution of theresulting radiometal complex of the conjugate. Thus, eg. theintroduction of ether groups in the linker will help to minimise plasmaprotein binding. Preferred linker groups -(A)_(n) have a backbone chainof linked atoms which make up the -(A)_(n) moiety contain 2 to 10 atoms,most preferably 2 to 5 atoms, with 2 or 3 atoms being especiallypreferred. A minimum linker group backbone chain of 2 atoms confers theadvantage that the chelator is well-separated from the biologicaltargeting moiety so that any interaction is minimised. A furtheradvantage is that the potential chelate ring size of the X and Z groupsis so large (at least 8 for a 2 atom backbone chain) that these groupsare unlikely to compete effectively with the coordination of thechelator to a radiometal. In this way, both the biological targetingcharacteristics of the biological targeting moiety, and the metalcomplexing capability of the diaminedioxime chelator is maintained inconjugates of this type.

Non-peptide linker groups such as alkylene groups or arylene groups havethe advantage that there are no significant hydrogen bondinginteractions with the conjugated biological targeting moiety so that thelinker does not wrap round onto the biological targeting moiety.Preferred alkylene spacer groups are —(CH₂)_(n)— where n is 2 to 5.Preferred arylene spacers are of formula:

-   -   where: a and b are independently 0, 1 or 2.

A preferred Y group is thus —CH₂CH₂—X-Z, most preferably —CH₂CH₂—NR⁴-Z,with Y=—CH₂CH₂—NH-Z being especially preferred. This grouping has theadditional advantage that it stems from the intermediateR³C(CH₂CH₂NH₂)₃, preferably the intermediate HC(CH₂CH₂NH₂)₃, which beingsymmetrical are much easier to synthesise, since triamines havingdifferent chain lengths would require the use of synthetic strategies tochemically distinguish the various amines (eg. via protecting groups).

The group X is a functional group which permits facile conjugation ofthe chelating agent to the biological targeting moiety Z. Since mostpeptides and proteins have available carboxyl or amino sites forfunctionalisation, preferred X groups when Z is a peptide or protein are—NR⁴— and —CO₂—, since these permit facile conjugation via amide bonds,Cysteine-containing peptides and proteins may have free thiol groups,preferred X groups when Z is a cysteine-containing peptide or protein,are thiolphilic groups such as maleimide and acrylamide, since thesepermit facile conjugation via thioether bonds.

Preferred diaminedioxime chelators of the present invention aresymmetrical, ie. the two —CR² ₂R² ₂NHCR¹ ₂C(═N—OH)R¹ substituents on the—CY(R³)— moiety are chosen to be the same. This has the advantage that,the chelator does not contain a chiral centre, since such centres maygenerate diastereomeric radiometal complexes and possibly require thepurification of particular isomers.

The chelator conjugates of Formula II may optionally be used in acidsalt form, ie. where one or more amines of either the diaminedioximedonor set or the Y group are protonated with a biocompatible acid. Suchsalts may be obtained directly, eg. by HPLC purification employing suchacids in the mobile phase (eg. acetic or trifluoroacetic acid), or byaddition of the biocompatible acid to a solution of the chelatorconjugate. The salt form may be useful to aid purification (eg. viaprecipitation or recrystallisation), or may facilitate dissolution inaqueous media (after which the pH can be readily adjusted if necessary).

The chelator conjugates of the present invention can be prepared byreaction of a bifunctional chelate of Formula III with the biologicaltargeting moiety:

-   -   where:    -   each R¹, R² and R³ is independently an R group;    -   E is -(A)_(n)-J        -   where: J is a functional goup suitable for conjugation to Z;            -   -(A)_(n)- is a linker group where each A is                independently —CR₂—, —CR═CR—, —C≡C—, —NRCO—, —CONR—,                —SO₂NR—, —NRSO₂—, —CR₂OCR₂—, —CR₂SCR₂—, —CR₂NRCR₂—, a                C₄₋₈ cycloheteroakylone group, a C₄₋₈ cycloalkylene                group, a C₅₋₁₂ arylene group, a C₃₋₁₂ heteroarylene                group or a polyalkyleneglycol, polylactic acid or                polyglycolic acid moiety;            -   n is an integer of value 0 to 10;                each R group is independently H or C₁₋₁₀ alkyl, C₃₋₁₀                alkylaryl, C₂₋₁₀ alkoxyalkyl, C₁₋₁₀ hydroxyalkyl, C₁₋₁₀                fluoroalkyl, or 2 or more R groups, together with the                atoms to which they are attached form a carbocyclic,                heterocyclic, saturated or unsaturated ring.

By the term “functional group suitable for conjugation” is meant afunctional group which will react with a corresponding functional groupof Z (typically an amine, carboxyl or thiol group) to chemically linkthe diaminedioxime chelator to Z. Preferred such functional groupssuitable for conjugation are: —NR⁵R⁶, —CO₂M, —NCS, —NCO, —SM¹, —OM¹,maleimide or acrylamide, where R⁵ and R⁶ are independently an R group orP^(G); M is H, a cation, P^(G) or an active ester, M¹ is H or P^(G); andP^(G) is a protecting group. The cation is suitably a positively-chargedcounterion, such as a metal ion, ammonium (NH₄ ⁺) or quaternary ammoniumor phosphonium ion. Preferably, the cation is a biocompatible cation.The terms ‘biocompatible cation’, ‘active ester’ and ‘protecting group’are as defined below. When the functional group is —NR⁵R⁶, at least oneand preferably both of R⁵ and R⁶ is H.

By the term “protecting group” is meant a group which inhibits orsuppresses undesirable chemical reactions, but which is designed to besufficiently reactive that it may be cleaved from the functional groupin question under mild enough conditions that do not modify the rest ofthe molecule. After deprotection the group in question may be used toconjugate the bifunctional chelate of Formula III to the biologicaltargeting moiety.

Protecting groups are well known to those skilled in the art and aresuitably chosen from, when J is —NR⁵R⁶: Boc (where Boc istert-butyloxycarbonyl), Fmoc (where Fmoc is fluorenylmethoxycarbonyl),trifluoroacetyl, allyloxycarbonyl, Dde [i.e.1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl] or Npys (i.e.3-nitro-2-pyridine sulfenyl); and when J is —CO₂P^(G): methyl ester,tert-butyl ester, benzyl ester when J is —OP^(G), suitable protectinggroups are: acetyl, benzoyl, trityl (Trt) or tetrabutyldimethylsilyl.When J is —SP^(G), suitable protecting groups are: Trityl and4-methoxybenzyl. The use of further protecting groups are described in‘Protective Groups in Organic Synthesis’, Theorodora W. Greene and PeterG. M. Wuts, (John Wiley & Sons, 1991).

By the term “biocompatible cation” is meant a positively chargedcounterion which forms a salt with an ionised, negatively charged group,where said positively charged counterion is also non-toxic and hencesuitable for administration to the mammalian body, especially the humanbody. Examples of suitable biocompatible cations include: the alkalimetals (eg. sodium or potassium); the alkaline earth metals (eg. calciumor magnesium); and the ammonium ion. A preferred biocompatible cation issodium ion (Na⁺).

By the term “active ester” is meant an ester derivative of thecarboxylic acid which is designed to be a better leaving group, andhence permit more facile reaction with nucleophiles present on thebiological targeting moiety such as amines. Examples of suitable activeesters are: N-hydroxysuccinimide (NHS), pentafluorophenol,pentafluorothiophenol, para-nitrophenol and hydroxybenzotriazole.

Amine-functionalised chelators of Formula III (ie. J=—NR⁵R⁶ can thus beconjugated to the carboxyl group(s) of a biological targeting moiety,via amide bonds. This coupling can be carried out directly (eg. usingsolid phase peptide synthesis), or in the presence of a suitableactivating agent, such as BOP [ie.benzotriazol-1-yloxy-tris(dimethylamino)-phosphonium] orN,N′-dicyclohexylcarbodiimide (DCCI), The coupling can also be carriedout via appropriate intermediates as is known in the art, such asactivated esters of the carboxyl group of the biological targetingmoiety. Alternatively, the pendant amine group of the bifunctionalchelator can first be converted to an isothiocyanate (—NCS) orisocyanate group (—NCO) group, which pet conjugation to amine-containingbiological targeting moieties, via the formation of thiourea and urealinkages respectively. Alternatively, the pendant amine group of thebifunctional chelator can be reacted with a diacid to introduce aterminal carboxyl group via a linker group. A bifunctional chelatorbearing a carboxyl function (ie. J=—CO₂M) can be used in a similarmanner to couple directly to amine-containing biological targetingmoieties via an amide bond. The bifunctional chelate may also bear agroup designed to react with thiol groups on the biological targetingmoiety to form stable thioether linkages. Examples of such groups to aremaleimides (which may be prepared by reaction of maleic anhydride withthe corresponding amine, followed by heating with acetic anhydride), andacrylamides (which may be prepared by reaction of acrylyl chloride withthe amine).

In a second aspect, the present invention provides radiometal complexesof the chelator conjugate described above. Suitable radiometals can beeither positron emitters such as ⁶⁴Cu, ⁴⁸V, ⁵²Fe, ⁵⁵Co, ^(94m)Tc or⁶⁸Ga; or γ-emitters such as ^(99m)Tc, ¹¹¹In, ^(113m)In or ⁶⁷Ga. Mostpreferred radiometals for diagnostic imaging are γ-emitters, especially^(99m)Tc. Metal complexes of certain radionuclides may be useful asradiopharmaceuticals for the radiotherapy of various diseases such ascancer or the treatment of thrombosis or restenosis. Usefulradioisotopes for such radiotherapeutic applications include: ⁹⁰Y, ⁸⁹Sr,⁶⁷Cu, ¹⁰³Pd, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁶⁹Er, ¹⁵³Sm and ¹⁹⁸Au. It is strongly preferthat the biological targeting moiety Z is bound to the chelator in sucha way that the linkage does not undergo facile metabolism in blood,which would result in the metal complex being cleaved off before thelabelled biological targeting moiety reaches the desired in vivo targetsite. The biological targeting moiety is therefore preferably covalentlybound to the metal complexes of the present invention via linkages whichare not readily metabolised (as are eg. ester linkages).

Preferred radiometal complexes of the present invention are symmetrical,ie. the two —CR² ₂R² ₂NHCR¹ ₂C(═N—OH)R¹ substituents on the —CY(R³)—moiety are chosen to be the same. This has the advantage that, theradiometal complex does not contain a chiral centre since such centresmay generate diastereomeric radiometal complexes and possibly requirethe purification of particular Isomers. It is also preferred that theradiometal complex of the chelator conjugate is electrically neutral.

It is believed that the ^(99m)Tc complexes of the chelators of thepresent invention are

neutral, Tc(V) dioxo complexes as shown above.

In the ^(99m)Tc-diaminedioxime complexes of the present invention, R³ ispreferably H. It is also preferred that at least one R² group is H, morepreferably all the R² groups are H. Each R¹ is preferably C₁₋₃ alkyl,C₂₋₄ alkoxyalkyl, C₁₋₃ hydroxyalkyl, or C₁₋₃ fluoroalkyl, and is mostpreferably C₁₋₃ alkyl or C₁₋₃ fluoroalkyl. It is most especiallypreferred that all the R¹ groups are CH₃. Preferred Y groups for the^(99m)Tc complex are as described above for the chelator conjugate.

Preferred radiometal complexes of the present invention wherein 2 ormore R groups which, together with the atoms to which they are attached,form a carbocyclic, heterocyclic, saturated or unsaturated ring,comprise such rings having 3- to 6-members, especially 5- or 6- members.Most preferred such rings are saturated carbocyclic rings. Preferredcarbocyclic rings are those in which 2 R¹ groups attached to either thesame or adjacent carbon atoms are combined to form 3- to 6-membered,especially 5- or 6-membered saturated rings.

The radiometal complexes of the present invention may be prepared byreacting a solution of the radiometal in the appropriate oxidation statewith the chelate conjugate at the appropriate pH. The solution maypreferably contain a ligand which complexes weakly to the metal (such asgluconate or citrate) i.e. to radiometal complex is prepared by ligandexchange or transchelation. Such conditions are useful to suppressundesirable side reactions such as hydrolysis of the metal ion. When theradiometal ion is ^(99m)Tc, the usual starting material is sodiumpertechnetate from a ⁹⁹Mo generator. Technetium is present in^(99m)Tc-pertechnetate in the Tc(VII) oxidation state, which isrelatively unreactive. The preparation of technetium complexes of loweroxidation state Tc(I) to Tc(V) therefore usually requires the additionof a suitable pharmaceutically acceptable reducing agent such as sodiumdithionite, sodium bisulphite, ascorbic acid, formamidine sulphinicacid, stannous ion, Fe(II) or Cu(I), to facilitate complexation. Thepharmaceutically acceptable reducing agent is preferably a stannous saltmost preferably stannous chloride, stannous fluoride or stannoustartrate.

In a third aspect, the present invention provides radiopharmaceuticalswhich comprise the above radiometal complexes of the chelator conjugatesin a sterile form suitable for human administration. Suchradiopharmaceuticals are suitably supplied in either a container whichis provided with a seal which is suitable for single or multiplepuncturing with a hypodermic needle (e.g. a crimped-on septum sealclosure) whilst maintaining sterile integrity. Such containers maycontain single or multiple patient doses. Preferred multiple dosecontainers comprise a single bulk vial (e.g. of 10 to 30 cm³ volume)which contains multiple patient doses, whereby single patient doses canthus be withdrawn into clinical grade syringes at various time intervalsduring the viable lifetime of the preparation to suit the clinicalsituation. Prefilled syringes are designed to contain a single humandose, and are therefore preferably a disposable or other syringesuitable for clinical use. The pre-filled syringe may optionally beprovided with a syringe shield to protect the operator from radioactivedose. Suitable such radiopharmaceutical syringe shields are known in theart and preferably comprise either lead or tungsten.

A ^(99m)Tc radioactivity content suitable for a diagnostic imagingradiopharmaceutical is in the range 180 to 1500 MBq, depending on thesite to be imaged in vivo, the uptake and the target to backgroundratio. For heart imaging with a ^(99m)Tc radiopharmaceutical, ca. 1110MBq (30 mCi) may be used for a stress study, and ca. 350 MBq (10 mCi)for a rest study.

In a fourth aspect, the present invention provides non-radioactive kitsfor the preparation of the ^(99m)Tc radiopharmaceutical composition.Such kits are designed to give sterile radiopharmaceutical productssuitable for human administration, e.g. via direct injection into thebloodstream. For ^(99m)Tc, the kit is preferably lyophilised and isdesigned to be reconstituted with sterile ^(99m)Tc-pertechnetate (TcO₄⁻) from a ^(99m)Tc radioisotope generator to give a solution suitablefor human administration without further manipulation. Suitable kitscomprise a container (eg. a septum-sealed vial) containing the chelatorconjugate of Formula II in either free base or acid salt form, togetherwith a pharmaceutically acceptable reducing agent such as sodiumdithionite, sodium bisulphite, ascorbic acid, formamidine sulphinicacid, stannous ion, Fe(II) or Cu(I). The pharmaceutically acceptablereducing agent is preferably a stannous salt such as stannous chlorideor stannous tartrate. Alternatively, the kit may optionally contain ametal complex which, upon addition of the radiometal, undergoestransmetallation (i.e. metal exchange) giving the desired product.

The non-radioactive kits may optionally further comprise additionalcomponents such as a transchelator, radioprotectant, antimicrobialpreservative, pH-adjusting agent or filler. The “transchelator” is acompound which reacts rapidly to form a weak complex with technetium,then is displaced by the diaminedioxime. This minimises the risk offormation of reduced hydrolysed technetium (RHT) due to rapid reductionof pertechnetate competing with technetium complexation. Suitable suchtranschelators are salts of a weak organic acid, ie. an organic acidhaving a pKa in the range 3 to 7, with a biocompatible cation. Suitablesuch weak organic acids are acetic acid, citric acid, tartaric acid,gluconic acid, glucoheptonic acid, benzoic acid, phenols or phosphonicacids Hence, suitable salts are acetates, citrates, tartrates,gluconates, glucoheptonates, benzoates, phenolates or phosphonates.Preferred such salts are tartrates, gluconates, glucoheptonates,benzoates, or phosphonates, most preferably phosphonates, mostespecially diphosphonates. A preferred such transchelator is a salt ofMDP, ie. methylenediphosphonic acid, with a biocompatible cation.

By the term “radioprotectant” is meant a compound which inhibitsdegradation reactions, such as redox processes, by trappinghighly-reactive free radicals, such as oxygen-containing free radicalsarising from the radiolysis of water. The radioprotectants of thepresent invention are suitably chosen from: ascorbic acid,para-aminobenzoic acid (ie. 4-aminobenzoic acid), gentisic acid (ie.2,5-hydroxybenzoic acid) and salts thereof with a biocompatible cationas described above.

By the term “antimicrobial preservative” is meant an agent whichinhibits the growth of potentially harmful micro-organisms such asbacteria, yeasts or moulds. The antimicrobial preservative may alsoexhibit some bactericidal properties, depending on the dose. The mainrole of the antimicrobial preservative(s) of the present invention is toinhibit the growth of any such micro-organism in the radiopharmaceuticalcomposition post-reconstitution, ie. in the radioactive diagnosticproduct itself. The antimicrobial preservative may, however, alsooptionally be used to inhibit the growth of potentially harmfulmicro-organisms in one or more components of the non-radioactive kit ofthe present invention prior to reconstitution. Suitable antimicrobialpreservative(s) include: the parabens, ie. methyl, ethyl, propyl orbutyl paraben or mixtures thereof; benzyl alcohol; phenol; cresol;cetrimide and thiomersal. Preferred antimicrobial preservative(s) arethe parabens.

The term “pH-adjusting agent” means a compound or mixture of compoundsuseful to ensure that the pH of the reconstituted kit is withinacceptable limits (approximately pH 4.0 to 10.5) for human or mammalianadministration. Suitable such pH-adjusting agents includepharmaceutically acceptable buffers, such as tricine, phosphate or TRIS[ie. tris(hydroxymethyl)aminomethane], and pharmaceutically acceptablebases such as sodium carbonate, sodium bicarbonate or mixtures thereof.When the conjugate of Formula II is employed in acid salt form, the pHadjusting agent may optionally be provided in a separate vial orcontainer, so that the user of the kit can adjust the pH as part of amulti-step procedure.

By the term “filler” is meant a pharmaceutically acceptable bulkingagent which may facilitate material handling during production andlyophilisation. Suitable fillers include inorganic salts such as sodiumchloride, and water soluble sugars such as sucrose, maltose ortrehalose.

In a fifth aspect the present invention provides bifunctionaldiaminedioxime chelators of Formula III useful to preparechelator-biological targeting moiety conjugates:

-   -   where:    -   each R¹, R² and R³ is independently an R group;    -   E is -(A)_(n)-J        -   where: J is a functional group suitable for conjugation to            Z;            -   -(A)_(n)- is a linker group where each A is                independently —CR₂—, —CR═CR—, —C≡C—, —NRCO—, —CONR—,                —SO₂NR—, —NRSO₂—, —CR₂OCR₂—, —CR₂SCR₂, —CR₂NRCR₂—, a                C₄₋₈ cycloheteroalylene group, a C₄₋₈ cycloalkylene                group, a C₅₋₁₂ arylene group, a C₃₋₁₂ heteroarylene                group or a polyalkyleneglycol, polylactic acid or                polyglycolic acid moiety;            -   a is an integer of value 0 to 10;    -   each R group is independently H or C₁₋₁₀ alkyl, C₃₋₁₀ alkylaryl,        C₂₋₁₀ alkoxyalkyl, C₁₋₁₀ hydroxyalkyl, C₁₋₁₀ fluoroalkyl, or 2        or more R groups, together with the atoms to which they are        attached form a carbocyclic, heterocyclic, saturated or        unsaturated ring.

By the term “functional group suitable for conjugation” is meant afunctional group which will react with a corresponding functional groupof Z (typically an amine, carboxyl or thiol group) to chemically linkthe diaminedioxime chelator to Z. Preferred such functional groupssuitable for conjugation are: —NR⁵R⁶, —CO₂M, —NCS, —NCO, —SM¹, —OM¹,maleimide or acrylamide, where R⁵ and R⁶ are independently an R group orP^(G); M is H, a cation, P^(G) or an active ester M¹ is H or P^(G); andP^(G) is a protecting group. The cation is suitably a positively-chargedcounterion, such as a metal ion, ammonium (NH₄ ⁺) or quaternary ammoniumor phosphomium ion. Preferably, the cation is a biocompatible cation.The terms ‘biocompatible cation’, ‘active ester’ and ‘protecting group’are as defined above. When the functional group is —NR⁵R⁶, at least oneand preferably both of R⁵ and R⁶ is H.

In the bifunctional chelators of Formula III of the present invention,R³ is preferably H. It is also preferred that at least one R² group isH, more preferably all the R² groups are H. Each R¹ is preferably C₁₋₃alkyl, C₂₋₄ alkoxyalkyl, C₁₋₃ hydroxyalkyl, or C₁₋₃ fluoroalkyl, and ismost preferably C₁₋₃ alkyl or C₁₋₃ fluoroalkyl. It is most especiallypreferred that all the R¹ groups are CH₃.

Preferred bifunctional chelators wherein 2 or more R groups which,together with the atoms to which they are attached, form a carbocyclic,heterocyclic, saturated or unsaturated ring, comprise such rings having3- to 6-members, especially 5- or 6-members. Most preferred such ringsare saturated carbocyclic rings. Preferred carbocyclic rings are thosein which 2 R¹ groups attached to either the same or adjacent cartonatoms are combined to form 3- to 6-membered, especially 5- or 6-memberedsaturated rings.

The chelator conjugates of Formula III may optionally be used in acidsalt form, ie. where one or more amines of either the diaminedioximedonor set or the Y group are protonated with a biocompatible acid. Suchsalts may be obtained directly, eg. by HPLC purification employing suchacids in the mobile phase (eg. acetic or trifluoroacetic acid), or byaddition of the biocompatible acid to a solution of the chelatorconjugate. The salt form may be useful to aid purification (eg. viaprecipitation or recrystallisation), or may facilitate dissolution inaqueous media (after which the pH can be readily adjusted if necessary).

Preferred linker groups -(A)_(n) of the bifunctional chelator have abackbone chain of linked atoms which make up the -(A)_(n) moiety contain2 to 10 atoms, most preferably 2 to 5 atoms, with 2 or 3 atoms beingespecially preferred. A minimum linker group backbone chain of 2 atomsconfers the advantage that, after conjugation, the chelator iswell-separated form the biological targeting moiety so that anyinteraction is minimised. A further advantage is that the potentialchelate ring size of the X and Z groups is so large (at least 8 for a 2atom backbone chain) that these groups are unlikely to competeeffectively with the coordination of the chelator to a radiometal.

Non-peptide linker groups such as alkylene groups or arylene groups havethe advantage that there are no significant hydrogen bondinginteractions with the conjugated biological targeting moiety so that thelinker does not wrap round onto the biological targeting moiety.Preferred alkylene spacer groups are —(CH₂)_(n)— where n is 2 to 5.Preferred arylene spacer; are of formula:

-   -   where: a and b are independently 0, 1 or 2.

A preferred E group is thus —CH₂CH₂-J, most preferably —CH₂CH₂—NHR⁵ or—CH₂CH₂—CO₂H or active esters thereof, with E=—CH₂CH₂—NH₂ be especiallypreferred. The acid can also be converted to a mixed anhydride eg. byreacting with isobutylchloroformate and base. The mixed anhydride alsoreacts with nucleophiles such as amines. The grouping E=—CH₂CH₂—NH₂ hasthe additional advantage that it stems from the intermediateR³C(CH₂CH₂NH₂)₃, preferably the intermediate HC(CH₂CH₂NH₂)₃, which beingsymmetrical is much easier to synthesise, since triamines havingdifferent chain lengths would require the use of synthetic strategies tochemically distinguish the various amines (eg. via protecting groups).

Preferred bifunctional diaminedioxime chelators of Formula III of thepresent invention are symmetrical, ie. the two —C(R²)₂(R²)₂NHCR¹₂C(═N—OH)R¹ substituents on the —CY(R³)— moiety are chosen to be thesame. This has the advantage that, the chelator does not contain achiral centre, since such centres may generate diastereomeric radiometalcomplexes and possibly require the purification of particular isomers.An especially preferred bifunctional diaminedioxime chelator has theformula:

Acid salts of this compound are also within the scope of the presentinvention.

The bifunctional diaminedioxime chelator of the present invention maysuitably be prepared by alkylation of a compound of Formula IV:

where A, J, R², R³ and n are as defined for Formula m above, witheither:

-   -   (i) the appropriate chloronitroso derivative Cl—C(R¹)₂—CH(NO)R¹;    -   (ii) an alpha-chloro oxime of formula Cl—C(R¹)₂—C(═NOH)R¹;    -   (iii) an alpha-bromoketone of formula Br—C(R¹)₂—C(═O)R¹ followed        by conversion of the diaminediketone product to the        diaminedioxime with hydroxylamine.

Route (i) is described by S. Jurisson et al [Inorg. Chem., 26, 3576-82(1987]. Chloronitroso compounds can be obtained by treatment of theappropriate alkene with nitrosyl chloride (NOCl) as is described inExample 3. Further synthetic details of chloronitroso compounds aregiven by: Ramalingam, K. et al Synth. Commun. (1995) 25(5) 743-52;Glaser et al J. Org Chem. (1996), 61(3), 1047-48; Clapp, Leallyn B.; etal J. Org Chem. (1971), 36(8) 1169-70; Saito, Giulichi et al ShizenKagaku (1995), 47, 41-9 and Schulz, Manfred Z. Chem (1981), 21(11),404-5. Route (iii) is described in broad terms by Nowotnik e al[Tetrahedron, 50(29), p.8617-8632 (1994)]. Alpha-chloro-oximes can beobtained by oximation of the corresponding alpha-chloro-ketone oraldehyde, which are commercially available. Alpha-bromoketones arecommercially available.

When J is —NH₂, the triamine of Formula IV may optionally first bemono-protected such that the J group primary amine is protected. Thediaminedioxime is then prepared according to routes (i), (ii) or (iii)above, then the protecting group is removed. Suitable protecting groupsare known in the art and include BOC (ie. tert-butoxycarbonyl) or Fmocas described above.

Compounds of Formula IV are suitably prepared from HC(CH₂CH₂OAc)₃ byhydrolysing one or more of the acetate esters to a primary alcohol(s)and converting to a leaving group such as a methanesulphonate ester withmethanesulphonyl chloride and pyridine. This leaving group may then bedisplaced with a suitable nucleophile that may be converted to thedesired functionality. To generate a carboxylic acid (ie. J=—CO₂H) acyanide anion would be used. Acid hydrolysis of the cyanide wouldgenerate the desired carboxylic acid. To generate an amine, an azidenucleophile would be used to generate an alkyl azide. Hydrogenation ofthe alkyl azide would produce at amine. To generate a thiol (ie. J=—SH),displacement of the leaving group with thioacetic acid anion would givea thioacetate which on acid hydrolysis would produce the thiol.

In a further aspect the present invention provides a compound of FormulaV:HC(CH₂CH₂NR⁷R⁸)₃,  Formula V

-   -   where R⁷ and R⁸ are independently H or P^(G), or R⁷ and R⁸        together form P^(G);        -   or a salt thereof.

P^(G) is a protecting group as defined above. The compounds of Formula Vare useful precursors to a range of bifunctional chelators of thepresent invention. A preferred compound of Formula V isHC(CH₂CH₂NH₂)₂(CH₂CH₂NR⁷R⁸), ie. a mono-protected triamine as describedabove. Most preferably, all the R⁷ and R⁸ groups are H, ie. the compoundHC(CH₂CH₂NH₂)₃, or an acid salt thereof, is a preferred compound of thepresent invention.

FIG. 1 shows the chemical structures of Compounds 1 to 6. FIG. 2 showsthe chemical structure of Compound 5 in full. FIG. 3 shows the reactionscheme for the azide synthesis of 1,1,1-tris(2-aminoethyl)amine ofExample 1. FIG. 4 shows the reaction scheme for the alternativesynthesis of 1,1,1-tris(2-aminoethyl)amine of Example 2.

The invention is illustrated by the non-limiting Examples detailedbelow. Example 1 describes the synthesis of the novel compound1,1,1-tris(2-aminoethyl)methane. Example 2 provides an alternativesynthesis of 1,1,1-tris(2-aminoethyl)methane which avoids the use ofpotentially hazardous azide intermediates. Example 3 describes thesynthesis of various chloronitrosoalkane precursors. Example 4 describesthe synthesis of a preferred amine-substituted bifunctionaldiaminedioxime of the present invention (Compound 1). Example 5describes the synthesis of a benzamide conjugate (Compound 2) ofCompound 1. Example 6 shows how a spacer group can be introduced whicheffectively converts the terminal amine function of the bifunctionalchelator to a terminal caboxyl function. Example 7 describes the solidphase synthesis of a thrombus-targeting peptide. Example 8 provides asynthesis of Compound 5, ie. a conjugate of Compound 1 with a targetingpeptide. Example 9 describes the synthesis of Compounds 7 and 8, whichare diaminedioxime analogues incorporating ring structures.

Example 10 compares the ^(99m)Tc-radiolabelling of Compound 2 with thatof the aza analogue prior art chelator (Compound 3), and shows that thechelators of the present invention give much more efficient and rapidlabelling under milder conditions, ie. room temperature and at lessalkaline pH. Thus, the prior art chelator requires pH 10 and a time ofat least 120 min at room temperature to give RCP's of over 80%, whereasCompound 3 labels at over 95% RCP within 15 minutes. Example 11 showsthat Compound 5 labels with ^(99m)Tc to give a high radiochemical puritypreparation. Example 12 shows that ^(99m)Tc-labelled Compound 5 showscomparable blood clot uptake in vitro to the prior art compound^(99m)Tc-Compound 6, and that hence the biological targeting propertiesof the peptide are retained when conjugated to the diaminedioximechelators of the present invention. Example 13 shows the ^(99m)Tcradiolabelling under mild conditions of Compound 9, which is a conjugateof Compound 1 with a cyclic peptide having relatively sensitivedisulphide bonds.

EXAMPLE 1 Synthesis of 1,1,1tris(2-aminoethyl)methane.

Step 1(a): 3(methoxycarbonylmethylene)glutaric acid dimethylester.

Carbomethoxymethylenetriphenylphosphorane (167 g, 0.5 mol) in toluene(600 ml) was treated with dimethyl 3-oxoglutarate (87 g, 0.5 mol) andthe reaction heated to 100° C. on an oil bath at 120° C. under anatmosphere of nitrogen for 36 h. The reaction was then concentrated invacuo and the oily residue triturated with 40/60 petrolether/diethylether 1:1, 600 ml. Triphenylphosphine oxide precipitatedout and the supernatant liquid was decanted/filtered off. The residue onevaporation in vacuo was Kugelrohr distilled under high vacuum Bpt (oventemperature 180-200° C. at 0.2 torr) to give3-(methoxycarbonylmethylene)glutaric acid dimethylester (89.08 g, 53%).

NMR ¹H(CDCl₃); δ 3.31 (2H, s, CH₂), 3.7(9H, s, 3×OCH₃), 3.87 (2H, s,CH₂), 5.79 (1H, s, ═CH₃) ppm.

NMR ¹³C(CDCl₃), δ 36.56,CH₃, 48.7, 2×CH₃, 52.09 and 52.5 (2×CH₂); 122.3and 146.16 C═CH; 165.9, 170.0 and 170.5 3×COO ppm.

Step 1(b): Hydrogenation of 3-(methoxycarbonylmethylene)glutaric aciddimethylester.

3-(methoxycarbonylmethylene)glutaric acid dimethylester (89 g, 267 mmol)in methanol (200 ml) was shaken with (10% palladium on charcoal: 50%water) (9 g) under an atmosphere of hydrogen gas (3.5 bar) for (30 h).The solution was filtered through kieselguhr and concentrated in vacuoto give 3-(methoxycarbonylmethyl)glutaric acid dimethylester as an oil,yield (84.9 g, 94%).

NMR ¹H(CDCl₃), δ 2.48 (6H, d, J=8 Hz, 3×CH₂), 2.78 (1H, hextet, J=8 HzCH, ) 3.7 (9H, s, 3×CH₃).

NMR ¹³C(CDCl₃), δ 28.6, CH; 37.50, 3×CH₃; 51.6, 3×CH₂; 172.28,3×COO.

Step 1(C): Reduction and esterification of trimethyl ester to thetriacetate.

Under an atmosphere of nitrogen in a 3 necked 2 L round bottomed flasklithium aluminium hydride (20 g, 588 mmol) in tetrahydrofuran (400 ml)was treated cautiously with tris(methyloxycarbonylmethyl)methane (40 g,212 mmol) in tetrahydrofluran (200 ml) over 1 h. A strongly exothermicreaction occurred, causing the solvent to reflux strongly. The reactionwas heated on an oil bath at 90° C. at reflux for 3 days. The reactionwas quenched by the cautious dropwise addition of acetic acid (100 ml)until the evolution of hydrogen ceased. The stirred reaction mixture wascautiously treated with acetic anhydride solution (500 ml) at such arate as to cause gentle reflux. The flask was equipped for distillationand stirred and then heating at 90° C. (oil bath temperature) to distilout the tetrahydrofuran. A further portion of acetic anhydride (300 ml)was added, the reaction returned to reflux configuration and stirred andheated in an oil bath at 140° C. for 5 hr. The reaction was allowed tocool and filtered. The aluminum oxide precipitate was washed with ethylacetate and the combined filtrates concentrated on a rotary evaporatorat a water bath temperature of 50° C. in vacuo (5 mmHg) to afford anoil. The oil was taken up in ethyl acetate (500 ml) and washed withsaturated aqueous potassium carbonate solution. The ethyl acetatesolution was separated, dried over sodium sulphate, and concentrated invacuo to afford an oil. The oil was Kugelrohr distilled in high vacuumto give tris(2-acetoxyethyl)methane (45.3 g, 95.9%) as an oil. Bp. 220°C. at 0.1 mmHg.

NMR ¹H(CDCl₃), δ 1.66(7H, m, 3×CH₂, CH), 2.08(1H, s, 3×CH₃); 4.1(6H, t,3×CH₂O).

NMR ¹³C(CDCl₃), δ 20.9, CH₃; 29.34, CH; 32.17, CH₂; 62.15, CH₂O; 171,CO.

Step 1(d); Removal of Acetate groups from the triacetate.

Tris(2-acetoxyethyl)methane (45.3 g, 165 mM) in methanol (200 ml) and880 ammonia (100 ml) was heated on an oil bath at 80° C. for 2 days. Thereaction was treated with a further portion of 880 ammonia (50 ml) andheated at 80° C. in an oil bath for 24 h. A further portion of 880ammonia (50 ml) was added and the reaction heated at 80° C. for 24 h.The reaction was then concentrated in vacuo to remove all solvents togive an oil. This was taken up into 880 ammonia (150 ml) and heated at80° C. for 24 h. The reaction was then concentrated in vacuo to removeall solvents to give an oil. Kugelrohr distillation gave acetamide bp170-180 0.2 mm. The bulbs containing the acetamide were washed clean andthe distillation continued. Tris(2-hydroxyethyl)methane (22.53 g, 92%)distilled at bp 220° C. 0.2 mm.

NMR ¹H(CDCl₃), δ 1.45(6H, q, 3×CH₂), 2.2(1H, quintet, CH); 3.7(6H, t3×CH₂OH); 5.5(3H, brs, 3×OH).

NMR ¹³C(CDCl₃), δ 22.13, CH; 33.95, 3×CH₂; 57.8, 3×CH₂OH.

Step 1(e) Conversion of the triol to the tris(methanesulphonate).

To an stirred ice-cooled solution of tris(2hydroxyethyl)methane (10 g,0.0676 mol) in dichloromethane (50 ml) was slowly dripped a solution ofmethanesulphonyl chloride (40 g, 0.349 mol) in dichloromethane (50 ml)under nitrogen at such a rate that the temperature did not rise above15° C. Pyridine (21.4 g, 0.27 mol, 4 eq) dissolved in dichloromethane(50 ml) was then added drop-wise at such a rate that the temperature didnot rise above 15° C., exothermic reaction. The reaction was left tostir at room temperature for 24 h and then treated with 5 N hydrochloricacid solution (80 ml) and the layers separated. The aqueous layer wasextracted with further dichloromethane (50 ml) and the organic extractscombined, dried over sodium sulphate, filtered and concentrated in vacuoto give tris[2-(methylsulphonyloxy)ethyl]methane contaminated withexcess methanesulphonyl chloride. The theoretical yield was 25.8 g.

NMR ¹H(CDCl₃), δ 4.3 (6H, t, 2×CH₂), 3.0 (9H, s, 3×CH₃), 2 (1H, hextet,CH), 1.85 (6H, q, 3×CH₂).

Step 1(f): Preparation of 1,1,1-tris(2-azidoethyl)methane.

A stirred solution of tris[2-(methylsulphonyloxy)ethyl]methane [fromStep 1(e), contaminated with excess methylsulphonyl chloride] (25.8 g,67 mmol, theoretical) in dry DNF (250 ml) under nitrogen was treatedwith sodium azide (30.7 g, 0.47 mol) portion-wise over 15 minutes. Anexotherm was observed and the reaction was cooled on an ice bath. After30 minutes, the reaction mixture was heated on an oil bath at 50° C. for24 h. The reaction became brown in colour. The reaction was allowed tocool, treated with dilute potassium carbonate solution (200 ml) andextracted three times with 40/60 petrol ether/diethylether 10:1 (3×150ml). The organic extracts were washed with water (2×150 ml), dried oversodium sulphate and filtered. Ethanol (200 ml) was added to thepetrol/ether solution to keep the triazide in solution and the volumereduced in vacuo to no less than 200 ml. Ethanol (200 ml) was added andreconcentrated in vacuo to remove the last traces of petrol leaving noless than 200 ml of ethanolic solution. The ethanol solution of triazidewas used directly in Step 1 (g).

CARE: DO NOT REMOVE ALL THE SOLVENT AS THE AZIDE IS POTENTIALLYEXPLOSIVE AND SHOULD BE KEPT IN DILUTE SOLUTION AT ALL TIMES.

Less than 0.2 ml of the solution was evaporated in vacuum to remove theethanol and an NMR run on this small sample:

NMR ¹H(CDCl₃), δ 3.35 (6H, t, 3×CH₂), 1.8 (1H, septet, CH,), 1.6 (6H, q,3×CH₂).

Step 1(g): Preparation of 1,1,1-tris(2-aminoethyl)methane.

Tris(2-azidoethyl)methane (15.06 g, 0.0676 mol), (assuming 100% yieldfrom previous reaction) in ethanol (200 ml) was treated with 10%palladium on charcoal (2 g, 50% water) and hydrogenated for 12 h. Thereaction vessel was evacuated every 2 hours to remove nitrogen evolvedfrom the reaction and refilled with hydrogen. A sample was taken for NMRanalysis to confirm complete conversion of the triazide to the triamine.Caution: unreduced azide could explode on distillation. The reaction wasfiltered through a celite pad to remove the catalyst and concentrated invacuo to give tris(2-aminoethyl)methane as an oil. This was furtherpurified by Kugelrohr distillation bp.180-200° C. at 0.4 mm/Hg to give acolourless oil (8.1 g, 82.7% overall yield from the triol).

NMR ¹(CDCl₃), 2.72 (6H, t, 3×CH₂N), 1.41 (H, septet, CH), 1.39 (6H, q,3×CH₂).

NMR ¹³C(CDCl₃), δ 39.8 (CH₂NH₂), 38.2 (CH₂.), 31.0 (CH).

EXAMPLE 2 Alternative Preparation of 1,1,1-tris(2-aminoethyl)methane.

Step 2(a): Amidation of trimethylester with p-methoxy-benzylamine.

Tris(methyloxycarbonylmethyl)methane [2 g, 8.4 mmol; prepared as in Step1(b) above] was dissolved in p-methoxy-benzylamine (25 g, 178.6 mmol).The apparatus was set up for distillation and heated to 120° C. for 24hrs under nitrogen flow. The progress of the reaction was monitored bythe amount of methanol collected. The reaction mixture was cooled toambient temperature and 30 ml of ethyl acetate was added, then theprecipitated triamide product stirred for 30 min. The triamide wasisolated by filtration and the filter cake washed several times withsufficient amounts of ethyl acetate to remove excessp-methoxy-benzylamine. After drying 4.6 g, 100%, of a white powder wasobtained. The highly insoluble product was used directly in the nextstep without further purifcation or characterization.

Step 2(b): Preparation of1,1,1-tris[2-(p-methoxybenzylamino)ethyl]methane.

To a 1000 ml 3-necked round bottomed flask cooled in a ice-water baththe triamide from step 2(a) (10 g, 17.89 mmol) is carefully added to 250ml of 1M borane solution (3.5 g, 244.3 mmol) borane. After completeaddition the ice-water bath is removed and the reaction mixture slowlyheated to 60° C. The reaction mixture is stirred at 60° C. for 20 hrs. Asample of the reaction mixture (1 ml) was withdrawn, and mixed with 0.5ml 5N HCl and left standing for 30 min. To the sample 0.5 ml of 50 NaOHwas added, followed by 2 ml of water and the solution was stirred untilall of the white precipitate dissolved. The solution was extracted withether (5 ml) and evaporated. The residue was dissolved in acetonitrileat a concentration of 1 mg/ml and analysed by MS. If mono- and diamide(M+H/z=520 and 534) are seen in the MS spectrum, the reaction is notcomplete. To complete the reaction, a further 100 ml of 1M borane THFsolution is added and the reaction mixture stirred for 6 more hrs at 60°C. and a new sample withdrawn following the previous sampling procedure.Further addition of the 1M borane in THF solution is continued asnecessary until there is complete conversion to the triamine.

The reaction mixture is cooled to ambient temperature and 5N HCl isslowly added, [CARE: vigorous foam formation occurs!], HCl is addeduntil no more gas evolution is observed. The mixture is stirred for 30min and then evaporated. The cake is suspended in aqueous NaOH solution(20-40%; 1:2 w/v) and sired for 30 minutes. The mixture is then dilutedwith water (3 volumes). The mixture was then extracted with diethylether(2 ×150 ml) [CARE: do not use halogenated solvents]. The combinedorganic phases were then washed with water (1×200 ml), brine (150 ml)and dried over magnesium sulphate. Yield after evaporation: 7.6 g, 84%as oil.

NMR ¹H(CDCl₃), δ: 1.45, (6H, m, 3×CH₂; 1.54, (1H, septet, CH); 2.60 (6H,t, 3×CH₂N); 3.68 (6H, s, ArCH₂); 3.78 (9H, s, 3×CH₃O); 6.94(6H, d,6×Ar). 7.20(6H, d, 6×Ar).

NMR ¹³C(CDCl₃), δ: 32.17,CH; 34.44, CH₂; 47.00, CH₂; 53.56, ArCH₂;55.25, CH₃O; 113.78, Ar; 129.29, Ar; 132.61; Ar; 158.60, Ar;

Step 2(c): Preparation of 1,1,1-tris(2-aminoethyl)methane.

1,1,1-tris[2(p-methoxybenzylamino)ethyl]methane (20.0 gram, 0.036 mol)was dissolved in methanol (100 ml) and Pd(OH)₂ (5.0 gram) was added. Themixture was hydrogenated (3 bar, 100° C., in an autoclave) and stirredfor 5 hours. Pd(OH)₂ was added in two more portions (2×5 gram) after 10and 15 hours respectively. The reaction mixture was filtered and thefiltrate was washed with methanol. The combined organic phase wasevaporated and the residue was distilled under vacuum (1×10⁻², 110° C.)to give 2.60 gram (50%) of 1,1,1-tris(2-aminoethyl)methane identicalwith the previously described Example 1.

EXAMPLE 3 Preparation of 3-chloro-3-methyl-2-nitrosobutane

A mixture of 2-methylbut-2-ene (147 ml, 1.4 mol) and isoamyl nitrite(156 ml, 1.16 mol) was cooled to −30° C. in a bath of cardice andmethanol and vigorously stirred with an overhead air stirrer and treateddropwise with concenrated hydrochloric acid (140 ml, 1.68 mol ) at sucha rate that the temperature was maintained below −20° C. This requiresabout 1 h as there is a significant exotherm and care must be taken toprevent overheating. Ethanol (100 ml) was added to reduce the viscosityof the slurry that had formed at the end of the addition and thereaction stirred at −20 to −10° C. for a further 2 h to complete thereaction. The precipitate was collected by filtration under vacuum andwashed with 4×30 ml of cold (−20° C.) ethanol and 100 ml of ice coldwater, and dried in vacuo to give 3-chloro-3-methyl-2-nitrosobutane as awhite solid. The ethanol filtrate and washings were combined and dilutedwith water (200 ml) and cooled and allowed to stand for 1 h at −10° C.when a further crop of 3-chloro-3-methyl-2-nitrosobutane crystallisedout. The precipitate was collected by filtration and washed with theminimum of water and dried in vacuo to give a total yield of3-chloro-3-methyl-2-nitrosobutane (115 g 0.85 mol, 73%) >98% pure byNMR.

NMR ¹H(CDCl₃), As a mixture of isomers (isomer], 90%) 1.5 d, (2H, CH₃),1.65 d, (4H, 2×CH₃), 5.85,q, and 5.95,q, together 1H. (isomer2, 10%),1.76 s, (6H, 2×CH₃), 2.07(3H, CH₃).

1-Chloro-1-(1-nitrosoethyl)cyclopentane was prepared in an analogousmanner from ethylidenecyclopentane (yield 55%) [J.Org.Chem, 36(8)p.1169-70].

1-Chloro-1-(1-nitrosoethyl)cyclohexane was prepared in an analogousmanner from ethylidenecyclohexane (yield 63%) [J.Org.Chem., 36(8)p.1169-70]. δ_(H) (CDCl₃; 270 MHz), 1.52 (3H, d J_(HH) 7 Hz, CH₃),1.48-2.20 (10H, m, CH₂×5), 5.96 (1H, q, J_(HH)7 Hz, CH).

1-Chloro-1-methyl-2-nitroso-cyclohexane was prepared in an analogousmanner from 1-methyl-1-cyclohexene (yield 57%) [Ind J. Chem Sect B(1978) 16B(10) 917-20, Z. Chem. (1981), 21(11) 404-5, J. Pract. Chem(1978) 320(3) 433-51]. δ_(H) (CDCl₃; 270 MHz), 1.41-2.28 (11H, m, CH₃,CH_(2×4)), 5.72-5.79 (1H, m, CH).

EXAMPLE 4 Synthesis of bis[N-(1,1-dimethyl-2-N-hydroxyiminepropyl)2-aminoethyl]-(2-aminoethyl)methane (Compound 1)

To a solution of tris(2-aminoethyl)methane (4.047 g, 27.9 mmol) in dryethanol (30 ml) was added potassium carbonate anhydrous (7.7 g, 55.8mmol, 2 eq) at room temperature with vigorous stirring under a nitrogenatmosphere. A solution of 3-chloro-3-methyl-2-nitrosobutane (7.56 g,55.8 mol, 2 eq) was dissolved in dry ethanol (100 ml) and 75 ml of thissolution was dripped slowly into the reaction mixture. The reaction wasfollowed by TLC on silica [plates run in dichloromethane, methanol,concentrated (0.88 sg) ammonia; 100/30/5 and the TLC plate developed byspraying with ninhydrin and heating]. The mono-, di- and tri-alkylatedproducts were seen with RF's increasing in that order. Analytical HPLCwas run using RPR reverse phase column in a gradient of 7.5-75%acetonitrile in 3% aqueous ammonia. The reaction was concentrated invacuo to remove the ethanol and resuspended in water (110 ml). Theaqueous slurry was extracted with ether (100 ml) to remove some of thetrialkylated compound and lipophilic impurities leaving the mono anddesired dialkylated product in the water layer. The aqueous solution wasbuffered with ammonium acetate (2 eq, 4.3 g, 55.8 mol) to ensure goodchromatography. The aqueous solution was stored at 4° C. overnightbefore purifying by automated preparative HPLC.

Yield (2.2 g 6.4 mmol, 23%).

Mass spec; Positive ion 10 V cone voltage. Found: 344; calculatedM+H=344.

NMR ¹H(CDCl₃), δ 1.24(6H, s, 2×CH₃), 1.3(6H, s, 2×CH₃), 1.25-1.75(7H, m,3×CH₂, CH), (3H, s, 2×CH₂), 2.58 (4H, m, CH₂N), 2.88(2H, t CH₂N₂), 5.0(6H, s, NH₂, 2×NH, 2×OH).

NMR ¹H ((CD₃)₂SO) δ1.1 4×CH; 1.29, 3×CH₂; 2.1 (4H, t, 2×CH₂);

NMR ¹³C((CD₃)₂SO), δ 9.0 (4×CH₃), 25.8 (2×CH₃), 31.0 2×CH₂, 34.6 CH₂,56.8 2×CH₂N; 160.3, C═N.

HPLC conditions: flow rate 8 ml/min using a 25 mm PRP column

A=3% ammonia solution (sp.gr=0.88) /water,

B=Acetonitrile

Time % B 0 7.5 15 75.0 20 75.0 22 7.5 30 7.5

Load 3 ml of aqueous solution per run, and collect in a time window of12.5-13.5 min.

EXAMPLE 5 The Preparation of Compound 2—the Benzamide Conjugate ofCompound 1

Compound 1 (0.5 g, 1.45 mmol) in dry acetonitrile (50 ml) andtriethylamine (150 mg, 1.45 mmol) under an atmosphere of nitrogen wascooled on an ice bath to 0° C. Benzoic anhydride (330 mg, 1.45 mmol) wasadded to the stirred reaction and allowed to warm to room temperatureand left to stir overnight. The acetonitrile was removed in vacuo andthe residue redissolved in (50 ml) dichloromethane, washed with aqueouspotassium carbonate (2×50 ml), separated and dried over sodium sulphate.The aqueous potassium carbonate solution was extracted withdichloromethane (2×50 ml), dried over sodium sulphate, and the combineddichloromethane extracts concentrated in vacuo to a gum. Analytical HPLCindicated fat the product was not as pure as required and the materialwas therefore purified by automated preparative HPLC, giving Compound 2.The product analysed as one spot on both TLC antd analytical HPLC.

HPLC conditions: flow rate of 8 ml/min using a 150 mm×25 mm PRP column;Sample loaded in 2 ml of 30% ethanol water per run.

A=3% ammonia solution (sp.gr=0.88)/water.

B=Acetonitrile

Time % B 0 7.5 15 75.0 20 75.0 22 7.5 30 7.5

The required product eluted at 15.25-16.5 min. The product solution wasevaporated in vacuo to give (304 mg, 0.68 mmol, 47%) of a colourlessglassy foam m.p. 55° C.

NMR ¹H(CDCl₃), 1.26 (12H, s, 4×CH₃), 1.43 (2H, m, CH₂), 1.57 (4H, m,CH₂), 1.75 (1H, m, CH), 1.823(6H, s, 2×CH₃), 2.58, (4H, m, 2×CH₂N),3.56(2H, m, CH₂NHCO), 6.95(1H, m, NHCO), 7.42(3H, m, 3×ArH) 7.79(2H, d,ArH).

NMR ¹³C(CDCl₃) 10.09, 25.7, 26.1, 28.5, 32.8, 33.3, 37.93, 57.57 127.0128.4, 131.4, 158.98, 168.15.

M/S C₂₄H₄₁N₅O₃ M+H=448 Found 448

RF 0.8 in 100:30:5/CH₂Cl₂:MeOH: 880 Ammonia, visualised with ninhydrin.

EXAMPLE 6 Synthesis of bis[(1,1-dimethyl-2-N-hydroxyiminepropyl)2-aminoethyl]-(2-(Glutarylamide)ethyl)methane [Compound 4: theglutarylamide derivative of Compound 1]

Compound 1 (0.5 g, 1.45 mmol) in dry acetonitrile (50 ml) andtriethylamine (150 mg, 1.45 mmol) under an atmosphere of nitrogen wascooled on an ice bath to 0° C. Glutaric anhydride (165 mg, 1.45 mmol)was added to the stirred reaction and allowed to warm to roomtemperature and left to stir overnight. The precipitate that formedovernight was collected by filtration and dried in vacuo to give animpure sample of the title compound (267 mg, 0.583 mmol, 40%). Thefiltrate was concentrated in vacuo to give a colourless glass whichtogether with the precipitate that had been collected was redissolved in5% 0.880 sg ammonia water (50 ml) and purified by automated preparativeHPLC. HPLC conditions: flow rate 8 ml/min, using a 150 mm×25 mm PRPcolumn Sample loaded in 2 ml of solution per run.

A=3% ammonia solution (sp.gr=0.88) /water.

B=Acetonitrile

Time % B 0 7.5 15 75.0 20 75.0 22 7.5 31 7.5

The required product eluted at 15.25-16.5 min. The product solution wasevaporated in vacuo to give (304 mg, 0.68 mmol 47%) of a colourlessglassy foam m.p. 54.8° C. The product analysed as one spot on both TLCand analytical HPLC.

NMR ¹H(DMSO), 0.7(12H, s, 4×CH₃), 0.85(4H, m, 2×CH₂), 1.0(1H, m, CH),1.3(6H, s, 2×CH₃), 1.3(4H, m, 2×CH₂), 1.6(2H, m, CH₂), 1.75 (6, m,3×CH₂), 2.6(2, m, , 2×OH) 3.2 (2H, t, NH) 7.3(1H, t, NH).

NMR ¹³C(CD₃SO) 8.97, 20.51, 20.91, 25.09, 25.60, 31.06, 33.41, 33.86,56.89, 66.99 160.07, 1712.34, 174.35 174.56

M/S C₂₂H₄₃N₅O₅ M+H=457 Found 457.6

EXAMPLE 7 Synthesis of the Protected Peptide Ac-NOEOVSP(3-I)YTLLKG

The protected peptideAc-Asn(Trt)-Gln(Trt)-Glu(OtBu)-Gln(Trt)-Val-Ser(tBu)-Pro-Tyr(3I)-Thr(tBu)-Leu-Leu-Lys(Boc)-Gly-OHwas assembled on a 2-chlorotrityl solid phase resin by anchoringFmoc-Gly- to the resin, and then successive deprotections/couplingcycles with the appropriate protected amino acids and the couplingreagents DCCI and HOBt. The terminal asparagine is acetylated, cleavedfrom the resin using 0.5% TFA and the peptide was used without furtherpurification.

EXAMPLE 8 Synthesis of Compound 5—A Peptide Conjugate of Compound 1

The protected Ac-NQEQVSPY(3I)TLLKG peptide of Example 7 was cleaved fromthe solid phase resin and then coupled with Compound 1 in solution usingBenzotriazole-1-yl-oxytris-pyrrolidino-phosphonium hexafluorophosphateand 1-hydroxybenzotriazole as the coupling agents. Compound 5 wasobtained by deprotection in reagent K (reagent K is 82.5% TFA, 5%phenol, 5% processed water, 5% thioanisole, 2.5% ethanedithiol). Thecrude peptide was first purified by RP-HPLC using TFA followed by asecond purification and salt exchange with acetic acid, lyophilisation,filtration with a 0.22μ filter and a final lyophilisation to giveCompound 5.

The prior art aza-diaminedioxime chelate conjugate (Compound 6—seeFIG. 1) of the same peptide, ie. Ac-NQEQVSPY(3I)TLLKG was prepared inthe same manner for comparison.

EXAMPLE 9 Peparation of1-(1-{3-(Z-Aminoethyl)-5-[1-(1-hydroxyliminoethyl)cyclohexylamino]pentylamino}cyclohexyl)ethanone dioxime [Compound 7]

To a solution of 1,1,1-tris(2-aminoethyl)methane (0.96 g, 6.6 mmol) indry ethanol (7.5 ml) was added potassium carbonate (anhydrous) (1.8 g,13 mmol) and triethylamine (1.33 g, 13 mmol) at room temperature withvigorous stirring under a nitrogen atmosphere. A solution of1-chloro-1-(1-nitrosoethyl)cyclohexane (2.3 g, 13 mmol) indichloromethane (30 ml) was added dropwise over 1 h. The mixture wasthen left to stir at room temperature for 18 h. The solvent was thenremoved under reduced pressure. Water (30 ml) and ether (25 ml) werethen added to the reaction residue. The aqueous phase and the organicphase were then separated.

HPLC:

ISOCRATIC: 90% B (MeOH) 10% (NH₃ 3%). Ether extract: HPLC showed twomajor bands-first band: dioxime, second band: trioxime. Dioxime: (0.55g, 20%), FAB m/z 424 (M+H), HRMS: Found: 424.3642, calc'd: 424.3652(C₂₃H₄₅N₅O₂).

NMR:

δ_(H) (CDCl₃; 270 MHz), 1.34-1.72 (33H, m, CH, CH₂×13, CH₃×2), 2.18-2.33(4H, m, NCH₂×2), 2.56-2.69 (2H, m, NCH₂).

The compound1-(1-{3-(2-aminoethyl)-5-[1-(1-hydroxyiminoethyl)cyclohexylamino]pentylamino}cyclohexyl)ethanonedioxime [Compound 8] was prepared in an analogous manner:

FAB m/z 396 (M+H), HRMS: Found: 396.3322, calc'd: 396.3339 (C₂₁H₄₂N₅O₃).

EXAMPLE 10 Comparative ^(99m)Tc Radiolabelling of Compound 2 vs theCorresponding Aza-Analogue (Compound 3, prior art)

A freeze-dried formulation containing:

-   -   23 μg Compound 2 (the benzamide derivative of Compound 1—see        Example3),    -   36 μg stannous chloride dihydrate,    -   90 μg Medronate trisodium,    -   4.0 mg Sodium acetate,        sealed under nitrogen gas (USP/NF) in a 10 mL glass vial was        prepared.

This was reconstituted with ^(99m)Tc-pertechnetate in saline from a^(99m)Tc generator, at room temperature and the RCP studied by HPLC andITLC (instant thin layer chromatography). The results were compared withthose of Compound 3, and are shown in Tables 1 and 2:

TABLE 1 ITLC radiochemical purity results (%): Time post- Compound 3Compound 3 reconstitution (prior art) (prior art) Compound 2 (min) pH 9pH 10 pH 9 15 13.2 37.0 96.5 12.8 36.4 30 28.4 55.3 96.3 24.3 53.4 6047.6 69.5 97.0 44.4 71.0 120 77.4 85.0 71.1 82.7

TABLE 2 ITLC and HPLC radiochemical purity results for Compound 2 (%):Compound 2. Time post- reconstitution (min) pH 9 15 ITLC 95 (5% RHT) 15HPLC 97.7 where RHT = reduced hydrolysed technetium.

EXAMPLE 11 ^(99m)Tc Labelling of Compound 5—A Peptide Conjugate ofCompound 1

A freeze-dried formulation containing:

-   -   50 μg PABA (para-aminobenzoic acid),    -   30 μg SnCl₂,    -   90 μg MDP (methylenediphosphonic acid),    -   1.32 mg NaHCO₃,    -   98 μg Na₂CO_(3,)    -   4 mg NaOAc,        was sealed under nitrogen gas in a 10 ml glass vial. The vial        was removed from freezer storage and left at room temperature        for 15 minutes, and was then reconstituted with 100 μl of a        solution containing Compound 5, ie. the peptide-chelator        conjugate        Ac-Asn-Gln-Glu-Gln-Val-Ser-Pro-(I-Tyr)-Thr-Leu-Leu-Lys-Gly-[Compound        1] (2 mg in 2 ml water) and Xml of ^(99m)Tc-pertechnetate in        saline, with a radioactive concentration of 0.5 GBq/ml, from an        Amertec II ^(99m)Tc generator at room temperature. The activity        was measured using an ion chamber. The RCP was measured using        ITLC and HPLC. The results are shown in Table 3 for different        values of X:

TABLE 3 ITLC and HPLC radiochemical purity results for Compound 5 (%):Reconstitution Time post- Volume Activity reconstitution RCP % RCP %Prep X (ml) (GBq) (min) (HPLC) (ITLC) 1 2 1.04 15 99.4 255 86.8 99.2 2 21 15 83.0 99.3 60 85.2 3 5 2.47 15 86.5 99.3 150 87.6 4 2 1.02 15 99.1140 86.6

EXAMPLE 12 In Vitro Clot Uptake of ^(99m)Tc-Labelled Compound 5 vs thatof ^(99m)Tc-Labelled Compound 6 (prior art)

The ^(99m)Tc radiolabelling was carried out according to Example 10.

Plasma (5 ml per test article) and thrombin (100 units ml⁻¹) wereremoved from storage (−20° C.) and allowed to defrost to roomtemperature. Plasma was observed prior to use to ensure that there wasno evidence of clot formation or sample degradation.

10 μl of ^(99m)Tc-Compound 5 or ^(99m)Tc-Compound 6 was added to one 5ml vial of plasma (rat, rabbit, dog and human). 10 μl of ^(99m)Tc-DTPAwas added to a second vial containing 5 ml of plasma in parallel as anegative control. Clot forming incubation mixtures were produced by theaddition of 800 μl of calcium tris buffer and 40 μl bovine thrombinsolution to four vials (calcium/thrombin rich clot forming buffer).Non-clot forming incubation, the background binding assay mixtures, wereproduced by the addition of 800 μl tris buffered saline solution to 40μl AnalaR water (calcium/thrombin deficient non-clot forming buffer).

400 μl of human plasma spiked with test article (^(99m)Tc-compound 5 or^(99m)Tc-Compound 6), or radiolabelled negative, control (^(99m)Tc-DTPA)were each added in quadruplicate to both of the calcium/thrombin richand calcium/thrombin deficient incubation mixtures. A singledefibrinating rod was added to each vial to facilitate plasma clotting.The assay vials were incubated at ambient temperature for 1 hour. Thereaction was terminated by the addition of 1 ml 0.4M EDTA solution toeach P7 vial.

The total radioactivity present was determined (in quadruplicate) byadding 400 μl samples of plasma previously spiked with test article andnegative controls into individual glass scintillation vials. Theradioactivity associated with these standards was determined by sodiumiodide scintigraphy. The contents of each P7 vial was decanted ontoindividual BSA blocked nitrocellulose filters over a vacuum manifold.Each P7 vial was rinsed with 2 ml TBST solution. Each filter was thenrinsed with four 5 ml washes of TBST solution. The clots were dried for1 hour over the vacuum manifold. The filter papers were then transferredto individual scintillation vials, and the radioactivity presentdetermined.

Non-specific binding of the test article to the nitrocellulose filterwas factored out by subtracting the total radioactivity present in theclot forming mixture from the total radioactivity present in thenon-clot forming mixture. The uptake into the clot alone (specific andnon-specific) was expressed as the percentage uptake of the test articlein the plasma by dividing the radioactivity present in the clot alone bythe average radioactivity present in the plasma standards thenmultiplying by 100:% uptake=% uptake into a clot, on a filter−% uptake on filter×100(background corrected)

The percentage specific binding was determined as the radioactive uptakethat was only due to Factor XIIIa formed isopeptide covalent bondsbetween fibrin and the test article. The specific binding was calculatedby subtracting the background (nitrocellulose filter) correctedpercentage uptake of the radiolabelled negative control (^(99m)Tc-DTPA)which bad no affinity for FXIIIa from the background (nitrocellulosefilter) corrected percentage uptake of the radiolabelled test article:Specific binding of test article=% uptake test article−% uptake DTPA(to clot) (in clot) (in clot)Effects on in vitro efficacy.

The data compared the uptake of ^(99m)Tc-Compound 5 and^(99m)Tc-Compound 6 into a forming plasma clot in vitro. There was nosignificant difference (p>0.05) in the efficacy of these two molecules(30.66±5.01 compared with 29.69±6.33) in this model of coagulation.

EXAMPLE 13 ^(99m)Tc-Labelling of Compound 9

Compound 9 is the conjugate of Compound 1 with the cyclic peptide shown,ie. [Compound 1]-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Cys-Tyr-OH.

Compound 9 was prepared in an analogous manner to Examples 7 and 8, andlabelled with ^(99m)Tc in solution (Preparation 1) or via a freeze-driedkit according to Example 10 (Preparation 2).

For Preparation 1, 100 μg of Compound 9 was dissolved in 1 ml of pH 8.5borate buffer. This was transferred to a P6 vial and sealed. 1 ml^(99m)Tc-pertechnetate in saline (1.0 GBq/ml, from an Amertec IIgenerator) was added at room temperature, together with 0.1 ml SnCl₂solution (10 mg SnCl₂ in 100 ml N₂ purged saline). The activity wasmeasured using an ion chamber. The RCP was measured using ITLC and HPLC.

Preparation 1 showed an RCP of 96% by ITLC, and Preparation 2 an RCP of82% by HPLC.

1. A chelator conjugate of the formula:


2. A radiometal complex of the chelator conjugate of claim
 1. 3. Theradiometal complex of claim 2, where the radiometal complex iselectrically neutral.
 4. The radiometal complex of claim 2, where theradiometal is ^(99m)Tc.
 5. A radiopharmaceutical which comprises theradiometal complex of claim 2, in a form suitable for humanadministration.
 6. A radiopharmaceutical of claim 5, where theradiometal is ^(99 m)Tc.
 7. A kit for the preparation of the ^(99m)Tcradiopharmaceutical of claim 6, which comprises: (i) the chelatorconjugate

a biocompatible reducing agent.
 8. The kit of claim 7, where thebiocompatible reducing agent is stannous.